Marek's Disease Herpesvirus Research Articles

Marek's disease herpesvirus transforming protein MEQ: a c-Jun analogue with an alternative life style.

In order to adapt to and to cope with an often hostile host environment, many viruses have evolved to encode products that are homologous to cellular proteins. These proteins exploit the existing host machinery and allow viruses to readily integrate into the host functional network. As a result, viruses are able to maneuver their journey seemingly effortlessly inside the host cell to achieve ultimate survival. Such molecular mimicries sometime go overboard, allowing viruses to overtake the cellular pathways or evade the immune system as do many of the retroviral oncogenes. Retroviral oncogenes are derived directly from host genes, and they are virtually identical to host genes in sequences except those mutations that make them unregulatable by host. Oncogenic herpesviruses also encode oncogenes, or transforming genes, which have independently evolved and are distantly related to host genes. However, these genes do share consensus structural motifs with cellular genes involved in cell growth and apoptosis and are functional analogues to host genes. The Marek's disease virus oncoprotein, MEQ, is one such example. MEQ is a basic region-leucine zipper (bZIP) transactivator which shares extensive homology with the Jun/Fos family of transcription factors within the bZIP domain, but not in other regions. Like all other bZIP proteins, MEQ is capable of dimerizing with itself and with a variety of bZIP partners including c-Jun, B-Jun, c-Fos, CREB, ATF-1, ATF-2, and SNF. MEQ-Jun heterodimers bind to a TRE/CRE-like sequence in the meq promoter region and have been shown to up-regulate MEQ expression in both chicken embryo fibroblasts and F9 cells. In addition, the bZIP and transactivation domains are interchangeable between MEQ and c-Jun in terms of transforming potential; i.e. MEQ can functionally substitute for c-Jun. These properties enable MEQ to engage in host cell processes by disguising itself as c-Jun. On the other hand, there are properties of MEQ notably different from c-Jun, which include its capability to bind RNA, to bind a CACAC-bent DNA structure as a homodimer, to inhibit apoptosis, and to interact with CDK2. MEQ's subcellular localization in the nucleolus and coiled body, is also different from Jun/Fos family of transactivators. These unique features may provide the MEQ with additional facility in regulating MDV replication, establishing latency, and cellular transformation. In this review, we will attempt to summarize the past research progress on MDV meq, with a focused on the similarities and differences between MEQ and cellular proteins, and between MEQ and other viral oncoproteins.

Atherosclerosis induced by infection with Marek’s disease herpesvirus in chickens

Background This research was suggested after crystals that we observed in herpesvirus-infected cell cultures were identified as cholesterol. Other reports and the development of defined reagents led us to select the use of Marek’s disease herpesvirus (MDV) infection of chickens to demonstrate a potential role of herpesviruses in the pathogenesis of atherosclerosis. Available for our use were a clone-purified strain of MDV of known virulence, genetically selected, specific pathogen–free chickens, and appropriate isolation facilities to design controlled experiments to fulfill Koch’s postulates. Methods and Results Experiments were performed to test the roles of both MDV and dietary cholesterol in atherosclerosis. The birds were examined 7 months after MDV infection with and without cholesterol feeding for gross and microscopic arterial lesions. Atherosclerotic lesions were found only in infected normocholesterolemic or hypercholesterolemic birds. The character and distribution of these lesions closely resembled those found in the chronic human arterial disease. Atherosclerotic lesions were not found in uninfected birds even if the birds were hypercholesterolemic. Conclusions Evidence was obtained from other experiments that after MDV infection, cholesterol and cholesteryl esters accumulated in cell cultures and in atherosclerotic lesions. These changes were associated with altered enzymatic activity of the cholesterol synthesis cycle. Immunization with turkey herpesvirus vaccine or SB-1 vaccine prevented atherosclerotic lesions. (Am Heart J 1999;138:S465-S468.)

Electron microscopic and immunohistochemical localization of Marek's disease (MD) herpesvirus particles in MD skin lymphomas.

Skin lymphomas induced in 11 specific-pathogen-free chickens by inoculation at 1 day of age with Marek's disease virus (MDV) were biopsied weekly and examined by electron microscopy and immunohistochemistry. In the sequentially biopsied lymphomas, immature MDV particles (abortive replication) were found only in the nuclei of necrotic lymphoblasts within necrotizing neoplasms. The necrotizing lymphomas were observed in two of the 11 experimental birds and were associated with prominent vascular endothelial cell injury, including fibrinoid necrosis of blood vessels. Nonnecrotizing lymphomas biopsied sequentially from the 11 experimental birds did not contain virus particles of any kind in the lymphoblasts and had no distinct vascular lesions. Immunohistochemically, MDV early antigen (pp38), but not late antigens (glycoproteins B and C), was detected only in the necrotizing lymphomas. These findings indicate that abortive MDV replication mainly occurred in necrotic lymphoblasts, which might have been induced by ischemia.

Multi-centric histiocytosis: Experimental induction in broiler and specific pathogen-free leghorn chickens

Seventy-five 3-day-old broiler chicks and twenty specific pathogen-free leghorn chicks were injected with 0.5 ml of a homogenate, prepared from organs from broilers diagnosed with naturally-occurring multicentric histiocytosis (MH). Equal numbers of uninjected broiler and leghorn chicks (controls) were maintained in adjacent pens. Ten weeks later, nine broilers had well-developed gross and microscopic MH lesions. The distribution and histological appearance of lesions in these experimental chicks was similar to lesions described in naturally occurring field cases. Six leghorns had gross lesions similar to those found in their broiler counterparts; however, in the leghorns, the cellular masses contained more lymphocytes and, additionally, masses were found in the gizzard musculature. One gizzard contained a sarcoma. Broiler chickens with MH weighed less than their control counterparts and were more likely to be anaemic. Sequences specific for reticuloendotheliosis viruses (REV) were found in the MH homogenate, in organs from most affected experimental leghorns and broilers, and in organs from a control broiler. However, REV were not isolated from these tissues, nor were specific antibodies for REV or avian leukosis/sarcoma viruses (ALV) found in chick serum. Leukosis/sarcoma viruses were isolated from some MH-affected experimental leghorns and broilers. Sequences specific for Marek's disease herpesvirus were not identified by polymerase chain reaction. The aetiology of MH remains unknown.

Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers.

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a beta-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek's disease herpesvirus. This was confirmed by characterization of additional 1.6kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages ( > 50%) of sequence identity to DNA polymerases of gamma-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine gamma-herpesviruses EHV-2 and EHV-5 with sequence identities of > 80%. This is a first indication that novel gamma-herpesviruses are present in wild and zoo equids.

Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek's disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek's disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Marek's disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

Open reading frame L1 of Marek's disease herpesvirus is not essential for in vitro and in vivo virus replication and establishment of latency.

Two mutant CV1988 Marek's disease virus (MDV) strains were developed in which a part of ORF L1 was replaced by lacZ with the SV40 early promoter. These mutant strains, CVIL1LacZ-A and -B, were inoculated into chickens to test the hypothesis that ORF L1 is involved in the induction and/or maintenance of latency. Mutant virus could be reisolated from lymphocytes obtained from chickens during both the lytic and latent phase of infection, indicating that ORF L1 is not essential for the induction and/or maintenance of latency or the reactivation from latency. Beta-galactosidase-positive lymphocytes were detected during the latent infection demonstrating that the SV40 early promoter can be active in recombinant MDV strains during latent infection. Although the insertion of lacZ was stable in cell culture, recombination within lacZ and the BamHI-L fragment was observed during in vivo infection.

Cytotoxic T lymphocyte response in chickens immunized with a recombinant fowlpox virus expressing Marek's disease herpesvirus glycoprotein B

Previously, we demonstrated that cytotoxic T lymphocytes (CTLs) from MHC: B 19B 19 and MHC: B 21B 21 chickens inoculated with a non-oncogenic Marek's disease virus (MDV) vaccine strain, SB-1/12 can lyse syngeneic reticuloendotheliosis virus (REV)-transformed cell lines expressing MDV pp38 or gB genes. In this study, we report the characterization of MDV gB-specific CTLs in chickens immunized with recombinant fowlpox virus expressing MDV gB gene (rFPV- gB). Spleen cells from rFPV- gB inoculated chickens (MHC: B 19B 19), depleted for CD4+, CD8+, TCR γ δ+, TCR α β1+ or TCR α β2+ cells were used as effector cells in chromium release assays. Effector cells depleted of CD8+ or TCR α β1+, but not CD4+, TCR γ δ+ or TCR α β2+ markedly reduced the percentage of specific release (%SR). Compared to the %SR caused by the SB-1/12-sensitized CTLs, the %SR caused by rFPV- gB-sensitized CTLs was low, but statistically significant. This is a first report on the induction of MDV gB-specific CD8+ CTLs in chickens immunized with rFPV- gB vaccine.

Phenotype analysis of lymphoid cells in Marek's disease of CD4+ or CD8+ T‐cell‐deficient chickens: Occurrence of double negative T‐cell tumour

Summary Chickens rendered either CD4+ or CD8+ T‐cell‐deficient by thymectomy and subsequent inoculation of anti‐CD4 or anti‐CD8 monoclonal antibody (mAb), were infected with Marek's disease herpesvirus (MDV). All the non‐treated control (4/4) and CD8‐deficient chickens (5/5) formed gross tumours. However, one of three CD4+ T‐cell‐deficient chickens developed gross tumours. Histologically, all chickens had tumourous lesions in the liver and nerve fibres. In CD4+ T‐cell‐deficient chickens, the phenotypes were CD3+CD4‐CD8‐Vß1‐αßTCR+ T‐cells and CD3‐CD4‐CD8‐ cells. These findings indicate that the double negative T‐cells (CD3+ CD4‐ CD8‐) and CD3‐ CD4‐ CD8‐ cells are transformed morphologically in addition to CD3+ CD4+ CD8‐ T‐cells by MDV infection.

Molecular evolution of infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1): An ancient example of the Alphaherpesviridae?

An analysis of two essential genes of infectious laryngotracheitis virus (ILTV), glycoprotein D (gD) and the immediate early gene, herpes simplex virus homologue ICP27, was performed with the equivalent gene homologues from several alphaherpesviruses. Amino acid (aa) sequence analysis revealed that these ILTV genes shared limited homology to other alphaherpesvirus equivalents and were distinct from the two other avian herpesviruses, Marek's disease virus (MDV) and herpesvirus of turkeys (HVT). Simplex and varicella group viruses are clearly separate from the avian group. The amino acid sequences of these ILTV genes will be presented with comparisions to the homologues from other alphaherviruses, contributing further evidence of the evolution of this group of viruses from a common progenitor and that ILTV could be an ancient example of the Alphaherpesvirinae.

Transactivation activity of Meq, a Marek's disease herpesvirus bZIP protein persistently expressed in latently infected transformed T cells

Marek's disease virus (MDV) is an avian herpesvirus that induces a variety of diseases, including T-cell lymphomas, in chickens. In latently infected, transformed lymphoid cells, very few viral transcripts or proteins are detected. We previously described a gene, meq (MDV EcoQ), which is persistently expressed in MDV-transformed tumor samples and cell lines. meq codes for a 339-amino-acid protein with a basic-leucine zipper domain near its N terminus and a proline-rich domain near its C terminus. The basic-leucine zipper domain shows homology with Jun/Fos family proteins, whereas the proline-rich domain resembles that of the WT-1 tumor suppressor protein. These structural features raise the possibility that Meq functions as a transcription factor in regulating viral latency or oncogenesis. In this report, we show that the proline-rich domain is a potent transcription activator when fused to the yeast (Saccharomyces cerevisiae) Gal4(1-147) DNA-binding domain. The transactivation activity maps to the C-terminal 130 amino acids, with the last 33 amino acids essential. In the absence of these 33 amino acids, a two-and-one-half proline-rich repeat structure was found to exhibit repression activity. We further show that Meq is able to dimerize not only with itself but also with c-Jun. Meq/c-Jun heterodimers bind to an AP1-like sequence in the meq promoter region with an affinity much greater than that of Meq/Meq or c-Jun/c-Jun homodimers. Cotransfection chloramphenicol acetyltransferase assays suggest that the Meq/c-Jun heterodimers can up-regulate Meq expression in both chicken embryo fibroblasts and F9 cells. Our data provide the first biochemical evidence that Meq is a transcriptional factor and identify c-Jun as one of Meq's interacting partners.

Efficacy of Marek's disease vaccines in Mhc heterozygous chickens: Mhc congenic x inbred line F1 matings.

The goal of this study is to demonstrate that Mhc (B) heterozygous chickens differ in efficacy of response to several Marek's disease (MD) vaccines. Four types of B2 heterozygotes, in addition to B2B2 homozygotes, were developed by crossing 15.B congenic males to inbred line 7(1) (B2B2) hens. The five types of F1 chicks were intermingled in isolators and vaccinated with one of four types of MD vaccine before inoculation with the very virulent Md5 strain of MD herpesvirus. The F1 chickens differ in development of protective immunity following MD vaccination from two perspectives. First, chickens of a particular Mhc genotype were protected better by some vaccines than others. Second, individual vaccine preparations protected some Mhc genotypes more effectively. We conclude that some MD vaccines are more appropriate than others for certain B-haplotypes when chickens are heterozygous for the Mhc. The value of using Mhc-congenic x inbred line F1 animals for studies concerning the influence of the Mhc on vaccinal immunity is discussed.

5252717 Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE: Leland F Velicer, Peter Brunovskis, Paul M Coussens assigned to Board of Trustees operating Michigan State University

5252717 Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE: Leland F Velicer, Peter Brunovskis, Paul M Coussens assigned to Board of Trustees operating Michigan State University

5252716 Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE: Leland F Velicer, Peter Brunovskis, Paul M Coussens assigned to Board of Trustees operating Michigan State University

5252716 Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE: Leland F Velicer, Peter Brunovskis, Paul M Coussens assigned to Board of Trustees operating Michigan State University

Culture Characterization and Viral Infection of Aortic Smooth Muscle Cells from Japanese Quail Susceptible to Atherosclerosis

Proliferation of aortic smooth muscle cells (SMCs), an important feature of atherosclerosis, occurs in the early stage of the disease in Japanese quail when fed an atherogenic diet. Quail aortic SMCs were isolated from a total of 32 quail and cultured in Eagle's minimum essential medium supplemented with 10% bovine fetal serum. Studies were carried out to characterize the cells in vitro. They grew actively in the early passages, but began to develop cytopathic changes after 3-4 passages and eventually cell death. The pathobiology had similarities to virus infected cells. Whole-mount electron microscopic examination detected virus-like particles in the cultured cells. All cell lines tested were positive for viral infection as screened by DNA dot blot hybridization using a genomic library of Marek's disease herpesvirus as a probe. However, the attempt to isolate the virus was unsuccessful. In summary, these cell lines are believed to be infected by a putative quail herpesvirus. Results of this in vitro cellular study support previous findings that the atherosclerosis susceptible quail are latently infected in vivo. Future studies will be needed to determine whether the virus plays a role in the pathogenesis of atherosclerosis in this animal model.

Enhanced Expression of the Marek's Disease Virus-Specific Phosphoproteins after Stable Transfection of MSB-1 Cells with the Marek's Disease Virus Homologue of ICP4

Phosphoprotein pp38, coded for by the BamHI-H fragment of the Marek's disease herpesvirus (MDV) genome is expressed in tumor cells and tumor cell lines. pp38 is associated with two other phosphoproteins, pp41 and pp24, and can be detected in a small percentage of tumor cells by indirect immunofluorescence assays (IIFA). The importance of MDV ICP4 for the regulation of pp38 expression was examined in the following MSB-1-derived cell lines stably transfected with the selection plasmid pNL1 [MDCC-CU221 (CU221)], pNL1 and the BamHI-A fragment of MDV DNA containing ICP4 (CU224), MDV ICP4 inserted in antisense direction in the eukaryotic expression vector pXT1 (CU222), or ICP4 in sense direction in pXT1 (CU223) or cotransfected with pNLI and EcoRl-linearized BamHI-A MDV DNA (CU225, -237, -243, -244). IIFA analysis showed that CU223 had a markedly increased expression of pp38, while CU224 had a slightly increased expression. No changes were noted in CU221 or CU222, while expression of pp38 was decreased in CU22,- 237,- 243, and 244. Radioimmunoprecipitation assays demonstrated that the expression of all three phosphoproteins was enhanced in CU223. Steady-state transcriptional analysis showed that CU223 had increased levels of pp38-specific (1,9 and 3.3 kb) and ICP4-specific (10.0 kb) transcripts.

Consistent chromosomal aberration in cell lines transformed with Marek's disease herpesvirus: evidence of genomic DNA amplification.

A specific chromosomal aberration was observed in 14 of 15 avian lymphoblastoid cell lines transformed with Marek's disease herpesvirus. This aberration, designated dup(1p)(p22-p23), appeared as an extra G-positive band and interband on the short arm of one chromosome I homolog. Using fluorescent in situ hybridization, we identified amplified genomic DNA sequences in this region. This amplification involves sequences linked to an endogenous retrovirus locus and genes in the histone multigene family. This aberration was not observed in cells transformed by reticuloendotheliosis virus or by avian leukosis virus, nor has it been observed in untransformed chicken cells. The induction of the 1p+ chromosomal aberration may be an essential event in the transformation of lymphocytes by Marek's disease virus.

Alterations in DNA sequence and RNA transcription of theBamHI-H fragment accompany attenuation of oncogenic Marek's disease herpesvirus

The nucleotide sequences of the BamHI-H fragment of the HPRS16 strain of Marek's disease virus (MDV) and of its attenuated derivative HPRS16/att have been determined. The results show that in addition to the tandem expansion of a 132 bp sequence from two copies in HPRS16 virus to eight copies in HPRS16/att, nucleotide substitutions, deletions, and insertions were also noted. Several potential open reading frames (ORFs) were identified. One of these (ORF 13) encoded a deduced protein, mol wt 32 kD, which is likely to be the serotype-1 specific phosphoprotein expressed in tumours (1) and mapped to an EcoRI fragment within BamHI-H (2). Our results suggest that this ORF is unlikely to be the B antigen (3). ORF 4, which had some similarity to CD4 and immunoglobulin M heavy chain, was encoded by a transcript that originated within the first copy of the 132 bp repeat. ORF 21, which mapped entirely within UL, encoded a deduced protein at least 322 amino acids long that had some similarity to varicella zoster virus (VZV) alpha trans-inducing protein. None of these ORFs was altered significantly by attenuation, except ORF 4 and another small ORF (ORF 3), 5' of the 132 bp repeats, which would probably fail to be transcribed because of truncation of an RNA transcript.

Antigen B of the vaccine strains of Marek's disease virus and herpesvirus of turkeys presents heat-labile group and serotype specific epitopes.

Antigen B of Marek's disease virus (MDV) vaccine strains CVI988 and SB1 (serotypes 1 and 2) and herpesvirus of turkeys (HVT) (serotype 3) is formed of oligomeric molecules that are detergent-stable and heat-labile. Immunoblots of native membranal extracts of HVT- and MDV-infected chick embryo fibroblasts (CEF) probed with avian monoserotypic antisera, murine monoclonal antibodies (mAb) to the three serotypes and mAb to antigen B showed two distinct patterns of high molecular weight oligomeric antigens. Serotypes 1 and 3 vaccine viruses formed one set and serotype 2, the other. Avian monotypic sera to serotypes 1 and 3 viruses detected two high molecular weight bands of 230 and > or = 300 kDa in MDV-1 and HVT-infected CEF but only a weak diffuse zone ranging from 130 to 230 kDa in extracts of SB1-infected CEF. No 300 kDa band was discernible in the SB1 extract when blotted with avian monotypic 1 and 3 antisera. MAbs to MDV serotypes 1 and 3 and to antigen B also detected the 230 and > or = 300 kDa antigens, while the mAb to SB1 detected a 50 kDa antigen in the SB1-infected extract only. Furthermore, the antigen B mAb did not reveal high mol. wt. oligomers in SB1-infected CEF extracts. Antigen B oligomers were rapidly destroyed by heating at 95 degrees C and the rate of denaturation of the 230 and > or = 300 kDa oligomers differed for each of the three vaccine viruses. We propose that antigen B of MDV1 and HVT has a complex conformation created by juxtaposition of dimers (230-250 kDa) and trimers (> or = 300 kDa), and is inserted in the infected cell membrane so that conformational, discontinuous epitopes are formed in addition to continuous epitopes. It appears that HVT protects chickens against oncogenic strains of MDV1 by virtue of the cross reactivity of the conformational determinants located on these oligomers. Serotype 2 vaccine shares some of its antigenic determinants with serotypes 1 and 3, while its unique immunogenic features form the basis of the protective synergism achieved when serotypes 2 and 3 vaccines are combined together.

Importance of Cell-Mediated Immunity in Marek's Disease and Other Viral Tumor Diseases

Cell-mediated immune (CMI) responses to viral tumor diseases are often used as examples of the importance of antiviral and antitumor immunity in chickens. Especially, reticuloendotheliosis virus (REV) and Marek's disease herpesvirus (MDV) are used as models to study the development of cytotoxic T-lymphocytes against viral and tumor antigens and activation of natural killer (NK) cells. Major histocompatibility complex Class I-restricted, antiviral cytotoxic T-lymphocytes expressing CD4–/CD8+ markers are induced after infection with REV. Thus far, this is the only example of Class I-restricted cytotoxic T-lymphocytes in chickens. Antiviral cytotoxic T-lymphocytes may be induced by infection with MDV or by vaccination, but conclusive evidence has not yet been provided. Antitumor responses have not been demonstrated against REV-induced tumors. Although Marek's disease is often used as an example for the importance of antitumor immunity, there is a lack of convincing data demonstrating antitumor immunity mediated by cytotoxic T-lymphocytes. Activation of NK cells by MDV infection or vaccination is probably an important part of CMI responses against Marek's disease viral antigens but not against tumor antigens.